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An interlaboratory collaborative study to validate a colorimetric phosphatase inhibition assay for quantitative determination of the okadaic acid (OA) toxins group in molluscs, OkaTest, was conducted. Eight test materials, including mussels, scallops, clams, and cockles, were analyzed as blind duplicates. Blank samples and materials containing different OA toxin levels ranging from 98 to 275 microg/kg OA equivalents were included. The study was carried out by a total of 16 laboratories from 11 different countries. Values obtained for repeatability relative standard deviations (RSDr) ranged from 5.4 to 11.2% (mean 7.5%). Reproducibility RSD (RSD(R)) values were between 7.6 and 13.2% (mean 9.9%). The Horwitz ratio (HorRat) values ranged between 0.4 and 0.6. A recovery assay was also carried out using a sample spiked with OA. A mean recovery of 98.0% and an RSD of 14.5% were obtained. The results obtained in this validation study indicate that the colorimetric phosphatase inhibition assay, OkaTest, is suitable for quantitative determination of the OA toxins group. OkaTest could be used as a test that is complementary to the reference method for monitoring the OA toxins group.

AOAC Official Method(SM) 2005.06 for the determination of saxitoxin (STX)-group toxins in shellfish by LC with fluorescence detection with precolumn oxidation was previously validated and adopted First Action following a collaborative study. However, the method was not validated for all key STX-group toxins, and procedures to quantify some of them were not provided. With more STX-group toxin standards commercially available and modifications to procedures, it was possible to overcome some of these difficulties. The European Union Reference Laboratory for Marine Biotoxins conducted an interlaboratory exercise to extend AOAC Official Method 2005.06 validation for dc-GTX2,3 and to compile precision data for several STX-group toxins. This paper reports the study design and the results obtained. The performance characteristics for dc-GTX2,3 (intralaboratory and interlaboratory precision, recovery, and theoretical quantification limit) were evaluated. The mean recoveries obtained for dc-GTX2,3 were, in general, low (53.1-58.6%). The RSD for reproducibility (RSD(r)%) for dc-GTX2,3 in all samples ranged from 28.2 to 45.7%, and HorRat values ranged from 1.5 to 2.8. The article also describes a hydrolysis protocol to convert GTX6 to NEO, which has been proven to be useful for the quantification of GTX6 while the GTX6 standard is not available. The performance of the participant laboratories in the application of this method was compared with that obtained from the original collaborative study of the method. Intralaboratory and interlaboratory precision data for several STX-group toxins, including dc-NEO and GTX6, are reported here. This study can be useful for those laboratories determining STX-group toxins to fully implement AOAC Official Method 2005.06 for official paralytic shellfish poisoning control. However the overall quantitative performance obtained with the method was poor for certain toxins.

An LC/MS/MS method has been developed, assessed, and intralaboratory-validated for the analysis of the lipophilic toxins currently regulated by European Union legislation: okadaic acid (OA) and dinophysistoxins 1 and 2, including their ester forms; azaspiracids 1, 2, and 3; pectenotoxins 1 and 2; yessotoxin (YTX), and the analogs 45 OH-YTX, Homo YTX, and 45 OH-Homo YTX; as well as for the analysis of 13-desmetil-spirolide C. The method consists of duplicate sample extraction with methanol and direct analysis of the crude extract without further cleanup or concentration. Ester forms of OA and dinophysistoxins are detected as the parent ions after alkaline hydrolysis of the extract. The validation process of this method was performed using both fortified and naturally contaminated samples, and experiments were designed according to International Organization for Standardization, International Union of Pure and Applied Chemistry, and AOAC guidelines. With the exception of YTX in fortified samples, RSDr below 15% and RSDR were below 25%. Recovery values were between 77 and 95%, and LOQs were below 60 microg/kg. These data together with validation experiments for recovery, selectivity, robustness, traceability, and linearity, as well as uncertainty calculations, are presented in this paper.

The application of ultra-performance rapid resolution LC on a 1.8 microm particle-size column coupled with tandem MS (RRLC-MS/MS) is described for the analysis of amnesic shellfish poisoning (ASP) toxins in shellfish. Complete resolution among domoic acid (DA) and the isomers was achieved in less than 3 min. The method was intralaboratory validated for direct analysis of crude extracts without further cleanup. It showed LODs ranging from 0.05 to 0.09 mg/kg and a working range that complied with the current regulatory level for DA of 20 mg/kg, and with the level of 4.5 mg/kg recently proposed by the European Food Safety Authority. Confirmatory capabilities were demonstrated according to the Commission Decision 2002/657/EC criteria. The results obtained by RRLC-MS/MS agreed with those provided by the reference LC-UV method, both intralaboratory for the analysis of blind samples (R2 = 0.9751) and interlaboratory through participation in the proficiency test for ASP toxins during 2009 (z-score = -0.962 and 0.177 for low- and high-contaminated samples, respectively). RRLC-MS/MS provided fast analysis and additional confirmatory capabilities for direct analysis of crude extracts while the performance and reliability of the results were maintained, even in very complex matrixes.